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The RNA-binding motif 45 (RBM45) protein accumulates in inclusion bodies in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with TDP-43 inclusions (FTLD-TDP) patients.

Collins M, Riascos D, Kovalik T, An J, Krupa K, Krupa K, Hood BL, Conrads TP, Renton AE, Traynor BJ, Bowser R - Acta Neuropathol. (2012)

Bottom Line: These RBM45 inclusions were observed in spinal cord motor neurons, glia and neurons of the dentate gyrus.By confocal microscopy, RBM45 co-localizes with ubiquitin and TDP-43 in inclusion bodies.In neurons containing RBM45 cytoplasmic inclusions we often detected the protein in a punctate pattern within the nucleus that lacked either TDP-43 or ubiquitin.

View Article: PubMed Central - PubMed

Affiliation: Departments of Neurobiology and Pathology, University of Pittsburgh, PA, USA.

ABSTRACT
RNA-binding protein pathology now represents one of the best characterized pathologic features of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration patients with TDP-43 or FUS pathology (FTLD-TDP and FTLD-FUS). Using liquid chromatography tandem mass spectrometry, we identified altered levels of the RNA-binding motif 45 (RBM45) protein in the cerebrospinal fluid (CSF) of ALS patients. This protein contains sequence similarities to TAR DNA-binding protein 43 (TDP-43) and fused-in-sarcoma (FUS) that are contained in cytoplasmic inclusions of ALS and FTLD-TDP or FTLD-FUS patients. To further characterize RBM45, we first verified the presence of RBM45 in CSF and spinal cord tissue extracts of ALS patients by immunoblot. We next used immunohistochemistry to examine the subcellular distribution of RBM45 and observed in a punctate staining pattern within nuclei of neurons and glia in the brain and spinal cord. We also detected RBM45 cytoplasmic inclusions in 91 % of ALS, 100 % of FTLD-TDP and 75 % of Alzheimer's disease (AD) cases. The most extensive RBM45 pathology was observed in patients that harbor the C9ORF72 hexanucleotide repeat expansion. These RBM45 inclusions were observed in spinal cord motor neurons, glia and neurons of the dentate gyrus. By confocal microscopy, RBM45 co-localizes with ubiquitin and TDP-43 in inclusion bodies. In neurons containing RBM45 cytoplasmic inclusions we often detected the protein in a punctate pattern within the nucleus that lacked either TDP-43 or ubiquitin. We identified RBM45 using a proteomic screen of CSF from ALS and control subjects for candidate biomarkers, and link this RNA-binding protein to inclusion pathology in ALS, FTLD-TDP and AD.

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Double-label immunofluorescence for RBM45 (green) and ubiquitin (red) in the spinal cord and hippocampus. a–c RBM45 co-localization with ubiquitin in ALS spinal cord motor neurons. DAPI visualizes nuclei (blue) in the merged images. Cytoplasmic ubiquitin inclusions are positive for RBM45; however, nuclear RBM45 is not labeled by ubiquitin (c). d, e Co-localization of RBM45 to ubiquitin cytoplasmic inclusions in the dentate gyrus. Arrows denote cells that exhibit co-localization of RBM45 and ubiquitin within an inclusion but retaining a speckled RBM45 nuclear immunostain. Note the lack of RBM45 labeling of an ubiquitin positive dystrophic neurite in the dentate gyrus as indicated by arrowhead (d). Scale bars denote 20 μm in panelsa–d and 30 μm in panele. Each panel represents the following case numbers in Table 1: a 10, b 7, c 22, d 27, e 26
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Fig7: Double-label immunofluorescence for RBM45 (green) and ubiquitin (red) in the spinal cord and hippocampus. a–c RBM45 co-localization with ubiquitin in ALS spinal cord motor neurons. DAPI visualizes nuclei (blue) in the merged images. Cytoplasmic ubiquitin inclusions are positive for RBM45; however, nuclear RBM45 is not labeled by ubiquitin (c). d, e Co-localization of RBM45 to ubiquitin cytoplasmic inclusions in the dentate gyrus. Arrows denote cells that exhibit co-localization of RBM45 and ubiquitin within an inclusion but retaining a speckled RBM45 nuclear immunostain. Note the lack of RBM45 labeling of an ubiquitin positive dystrophic neurite in the dentate gyrus as indicated by arrowhead (d). Scale bars denote 20 μm in panelsa–d and 30 μm in panele. Each panel represents the following case numbers in Table 1: a 10, b 7, c 22, d 27, e 26

Mentions: As ubiquitin-positive inclusions are a pathologic hallmark of both ALS and FTLD-TDP [14, 26], we examined the co-localization of RBM45 and ubiquitin-containing inclusions in ALS and FTLD-TDP. RBM45-containing cytoplasmic inclusions in lumbar spinal cord motor neurons of sporadic and familial ALS cases were frequently co-labeled with ubiquitin (Fig. 7a–c). RBM45 nuclear immunoreactivity in neurons or glia lacked ubiquitin staining (Fig. 7a, c). These results suggest that RBM45 pathology is highly coincident with ubiquitin in cytoplasmic inclusions, but nuclear RBM45 lacks ubiquitin.Fig. 7


The RNA-binding motif 45 (RBM45) protein accumulates in inclusion bodies in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with TDP-43 inclusions (FTLD-TDP) patients.

Collins M, Riascos D, Kovalik T, An J, Krupa K, Krupa K, Hood BL, Conrads TP, Renton AE, Traynor BJ, Bowser R - Acta Neuropathol. (2012)

Double-label immunofluorescence for RBM45 (green) and ubiquitin (red) in the spinal cord and hippocampus. a–c RBM45 co-localization with ubiquitin in ALS spinal cord motor neurons. DAPI visualizes nuclei (blue) in the merged images. Cytoplasmic ubiquitin inclusions are positive for RBM45; however, nuclear RBM45 is not labeled by ubiquitin (c). d, e Co-localization of RBM45 to ubiquitin cytoplasmic inclusions in the dentate gyrus. Arrows denote cells that exhibit co-localization of RBM45 and ubiquitin within an inclusion but retaining a speckled RBM45 nuclear immunostain. Note the lack of RBM45 labeling of an ubiquitin positive dystrophic neurite in the dentate gyrus as indicated by arrowhead (d). Scale bars denote 20 μm in panelsa–d and 30 μm in panele. Each panel represents the following case numbers in Table 1: a 10, b 7, c 22, d 27, e 26
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Related In: Results  -  Collection

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Fig7: Double-label immunofluorescence for RBM45 (green) and ubiquitin (red) in the spinal cord and hippocampus. a–c RBM45 co-localization with ubiquitin in ALS spinal cord motor neurons. DAPI visualizes nuclei (blue) in the merged images. Cytoplasmic ubiquitin inclusions are positive for RBM45; however, nuclear RBM45 is not labeled by ubiquitin (c). d, e Co-localization of RBM45 to ubiquitin cytoplasmic inclusions in the dentate gyrus. Arrows denote cells that exhibit co-localization of RBM45 and ubiquitin within an inclusion but retaining a speckled RBM45 nuclear immunostain. Note the lack of RBM45 labeling of an ubiquitin positive dystrophic neurite in the dentate gyrus as indicated by arrowhead (d). Scale bars denote 20 μm in panelsa–d and 30 μm in panele. Each panel represents the following case numbers in Table 1: a 10, b 7, c 22, d 27, e 26
Mentions: As ubiquitin-positive inclusions are a pathologic hallmark of both ALS and FTLD-TDP [14, 26], we examined the co-localization of RBM45 and ubiquitin-containing inclusions in ALS and FTLD-TDP. RBM45-containing cytoplasmic inclusions in lumbar spinal cord motor neurons of sporadic and familial ALS cases were frequently co-labeled with ubiquitin (Fig. 7a–c). RBM45 nuclear immunoreactivity in neurons or glia lacked ubiquitin staining (Fig. 7a, c). These results suggest that RBM45 pathology is highly coincident with ubiquitin in cytoplasmic inclusions, but nuclear RBM45 lacks ubiquitin.Fig. 7

Bottom Line: These RBM45 inclusions were observed in spinal cord motor neurons, glia and neurons of the dentate gyrus.By confocal microscopy, RBM45 co-localizes with ubiquitin and TDP-43 in inclusion bodies.In neurons containing RBM45 cytoplasmic inclusions we often detected the protein in a punctate pattern within the nucleus that lacked either TDP-43 or ubiquitin.

View Article: PubMed Central - PubMed

Affiliation: Departments of Neurobiology and Pathology, University of Pittsburgh, PA, USA.

ABSTRACT
RNA-binding protein pathology now represents one of the best characterized pathologic features of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration patients with TDP-43 or FUS pathology (FTLD-TDP and FTLD-FUS). Using liquid chromatography tandem mass spectrometry, we identified altered levels of the RNA-binding motif 45 (RBM45) protein in the cerebrospinal fluid (CSF) of ALS patients. This protein contains sequence similarities to TAR DNA-binding protein 43 (TDP-43) and fused-in-sarcoma (FUS) that are contained in cytoplasmic inclusions of ALS and FTLD-TDP or FTLD-FUS patients. To further characterize RBM45, we first verified the presence of RBM45 in CSF and spinal cord tissue extracts of ALS patients by immunoblot. We next used immunohistochemistry to examine the subcellular distribution of RBM45 and observed in a punctate staining pattern within nuclei of neurons and glia in the brain and spinal cord. We also detected RBM45 cytoplasmic inclusions in 91 % of ALS, 100 % of FTLD-TDP and 75 % of Alzheimer's disease (AD) cases. The most extensive RBM45 pathology was observed in patients that harbor the C9ORF72 hexanucleotide repeat expansion. These RBM45 inclusions were observed in spinal cord motor neurons, glia and neurons of the dentate gyrus. By confocal microscopy, RBM45 co-localizes with ubiquitin and TDP-43 in inclusion bodies. In neurons containing RBM45 cytoplasmic inclusions we often detected the protein in a punctate pattern within the nucleus that lacked either TDP-43 or ubiquitin. We identified RBM45 using a proteomic screen of CSF from ALS and control subjects for candidate biomarkers, and link this RNA-binding protein to inclusion pathology in ALS, FTLD-TDP and AD.

Show MeSH
Related in: MedlinePlus